EP0652963A1 - Cloning and/or sequencing vector. - Google Patents

Cloning and/or sequencing vector.

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Publication number
EP0652963A1
EP0652963A1 EP93915577A EP93915577A EP0652963A1 EP 0652963 A1 EP0652963 A1 EP 0652963A1 EP 93915577 A EP93915577 A EP 93915577A EP 93915577 A EP93915577 A EP 93915577A EP 0652963 A1 EP0652963 A1 EP 0652963A1
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Prior art keywords
nucleotide sequence
vector
protein
coding
poison
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EP93915577A
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German (de)
French (fr)
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EP0652963B1 (en
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Philippe Bernard
Philippe Gabant
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Universite Libre de Bruxelles ULB
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Universite Libre de Bruxelles ULB
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the invention relates to a cloning and / or sequencing vector allowing direct selection of recombinant clones.
  • the invention also relates to the prokaryotic cell transformed by this vector and the prokaryotic host cell of this vector as well as to the use of this cloning and sequencing vector for the selection and sequencing of recombinant clones.
  • Phage (M13 series) and plasmid (pUC series) cloning vectors containing many unique cloning sites were constructed by Messing et al (PNAS USA, 79, p 3642-3646 (1977), by Norrander et al ( Gene, 26, p 101-106 (1983) and Yanisch-perron et al (Gene, 33 p 103-119 (1985)).
  • the multiple cloning sites (MCS - multiple cloning sites) of these vectors are located in the coding sequence of the LacZ gene.
  • the discrimination between the transformed cells harboring a recombinant vector and the cells harboring a non-recombinant vector, is carried out by the technique of
  • Screening discrimination is based on the identification of a clone within a population of clones on the basis of a characteristic (color) which differentiates it. The selection does not require this characteristic, since by this method, we isolate only recombinant clones.
  • the screening process is based on the coloration of the recombinant clones (white coloration) and of the non-recombinant clones (blue coloration). This staining is based on the inactivation of the beta-galactosidase marker, preventing cleavage of the X-gal (5-bromo-4-chloro-3-indolyl-b-galactoside). Cell colonies harboring a non-recombinant vector produce a functional beta-galactosidase and by hydrolyzing the X-gal substrate produce a blue dye. The insertion of a DNA fragment into the Beta-galactosidase gene, generally prevents the cleavage of X-gal. The cells hosting a recombinant vector therefore have a white coloration.
  • the promoter of this vector comprises a region with several specific cloning sites (cleavage site for a restriction enzyme).
  • the sac B gene codes for levansucrase catalyzing the hydrolysis of sucrose into products toxic for E. coli; the direct selection of the mutants integrating a recombinant plasmid is done on a culture medium containing sucrose.
  • levansucrase is toxic, even in the absence of sucrose, it is therefore essential to suppress its synthesis if one wishes to obtain a large number of plasmid copies in the bacterial cytoplasm.
  • the impossibility of repressing the cytotoxic gene leads in the plasmid production phases, cell death and therefore a selective pressure towards mutated strains (characterized by an inactive lethal gene).
  • sucrose is often incorporated into bacterial culture media, it will be essential to prepare media completely free of sucrose to practice these manipulations.
  • Henrich et al (Gene, vol 42, n ° 3, 1986, p 345-349) describe a vector comprising the E gene derived from the bacteriophage ⁇ Xll ⁇ , said E gene being incorporated into the plasmid pUH84, under the control of the Lac promoter.
  • the E gene has six unique restriction sites (located throughout the E gene sequence) and codes for the gpE which causes cell lysis of E. coli.
  • the positive selection is carried out when a recombinant foreign gene has come to insert itself at one of the restriction sites.
  • this insertion of a foreign gene at a restriction site located in the sequence of the E gene, coding for gpE makes sequencing and / or PCR amplification of the foreign gene more difficult, because in this if it is sequenced, amplified and also characterized portions of unnecessary sequences belonging to the E gene coding for gpE.
  • Kuhn et al (Gene, vol 42, n ° 3, 1986, p 253-263) describe a vector comprising a large gene, coding for a restriction enzyme which kills by cleavage of the genome of the bacterium, said gene being incorporated in the plasmid pKG2 under the control of the LacUV5 promoter.
  • Cloning vectors of the state of the art have the disadvantage of having to be maintained in a host strain, containing the LacI repressor q episomally or repressor CI, to one inactivate the promoter prevent the expression of the killer gene and thereby cause the death of the host strain. Also, if it is desired to cause this strain to produce a large number of copies of the cloning vectors, the repressor will not be sufficient to prevent either a selective pressure modifying the cytotoxic activity of the vector, or a "genetic leakage", that is, the expression of certain copies of the vector and the death of the host strain.
  • a cloning vector which can incorporate large nucleotide fragments, which is easy to handle and which can be produced industrially by a microorganism; that is, it can be produced in large numbers of copies by a microorganism without causing its death.
  • AIMS OF THE INVENTION The present invention aims to provide a new cloning and / or sequencing vector, as well as its host strain, which are of easy and inexpensive construction and production, and which allow the direct selection of recombinant clones, without having the drawbacks of the aforementioned prior art.
  • a particular object of the present invention is to obtain a vector which allows specific and certain selection of the recombinant clones.
  • Another object of the present invention is to obtain a vector which allows the sequencing, amplification and / or characterization with the same primer, of any foreign DNA fragment (whatever its size) in recombinant clones .
  • Another object of the present invention is to obtain a vector which also allows easy extraction of this fragment of foreign DNA from the recombinant clone.
  • a final object of the present invention aims to obtain a host strain of said vector which makes it possible to obtain the production of a large number of copies of said vector without causing selection pressure modifying the cytotoxic activity of said vector or the death of the host strain.
  • Characteristic Elements of the Invention The invention relates to a new cloning and / or sequencing vector, comprising, incorporated into an autonomous replicating vector, at least one promoter nucleotide sequence and at least one nucleotide sequence coding for an active fusion protein as poison; said nucleotide sequence being obtained by the fusion of a coding nucleotide sequence comprising several unique cloning sites and of a nucleotide sequence coding for a poison protein.
  • the autonomously replicating vector is a virus or a plasmid such as a recombinant pUC plasmid.
  • the promoter nucleotide sequence can include any promoter, allowing expression of the nucleotide sequence encoding an active fusion protein as a poison.
  • this promoter nucleotide sequence is constituted by the promoter of the Lac operon.
  • the unique cloning sites (MCS) of the nucleotide sequence fused to the nucleotide sequence coding for the poison protein are absent from the rest of the nucleotide sequence of the vector according to the invention.
  • the nucleotide sequence of the gene coding for the poison protein comprises all or part of the nucleotide sequence of the wild-type gene coding for the protein CcdB.
  • the nucleotide sequence of the gene coding for the poison protein is devoid of the cleavage site of the restriction enzyme S al.
  • Another aspect of the invention relates to a prokaryotic cell transformed with the cloning vector according to the invention.
  • the invention also relates to a cell.
  • prokaryotic host of the vector according to the invention comprising a chromosomal I q , a high transformation rate and comprising a mutation conferring resistance to the poison activity of the fusion protein and / or comprising a gene coding for a poison protein of the fusion protein.
  • the prokaryotic host cell of the vector according to the invention comprises a mutation in the gene coding for the subunit A or in the gene coding for the subunit B of the gyrase and conferring resistance to the fusion protein and / or a gene encoding the CcdA protein poison from the fusion protein.
  • the prokaryotic cell is an Eschierichia coli cell comprising a mutation responsible for the substitution of arginine 462 by a cysteine in the amino acid sequence of the gyrase GyrA polypeptide, conferring resistance to the fusion protein.
  • this prokaryotic host cell also includes the Lacl q mutation.
  • the present invention also relates to fragments of the vector according to the invention, in particular primers for sequencing and / or amplification (for example by PCR) of the foreign nucleotide fragments inserted into the vector according to the invention.
  • these primers consist of sequences of 10 to 30 nucleotides which hybridize to the nucleotide sequences located on either side of the nucleotide sequence comprising several unique cloning sites, of the vector according to the invention.
  • a final aspect of the invention relates to the use of the vector according to the invention for the selection and sequencing of recombinant clones. Brief description of the figures
  • FIG. 1 schematically shows a cloning vector according to the present invention.
  • FIG. 2 shows the nucleotide sequence of the ccdB gene coding for the protein CcdB.
  • FIGS. 3 and 4 show the sequence nucleotide encoding the fusion protein of the cloning vectors pKIL18 and pKIL19 respectively. These sequences are provided with a nucleotide sequence containing multiple unique cleavage sites for different restriction enzymes. These pKIL18 and pKIL19 cloning vectors were obtained by in vitro recombination between the wild-type ccdB gene of plasmid F and the plasmids pUC18 and pUC19 respectively.
  • the cloning and / or sequencing vector 1 comprises incorporated into a self-replicating vector 2, at least one promoter nucleotide sequence 3 and at least one sequence nucleotide 4 encoding an active fusion protein as a poison; said nucleotide sequence 4 being obtained by the fusion of a coding nucleotide sequence 5 (or polylinker) comprising several (multiple) unique cloning sites (MCS) and of a nucleotide sequence 6 coding for a poison protein.
  • a coding nucleotide sequence 5 or polylinker
  • MCS multiple unique cloning sites
  • autonomous replicating vector 2 is intended to mean any nucleotide construct such as a virus or a plasmid (preferably a recombinant PUC series plasmid) capable of entering a microorganism, of recombining therein and / or of s 'replicate it.
  • FIG. 1 schematically represents a cloning vector according to the present invention, constructed from a plasmid of the pUC series (pUC18 and pUC19) described by Norrander et al (Construction of improved M13 vectors using oligodeoxinucleotide-directed mutagenesis, Gene, 26, p 101-106 (1983)) and by Yanisch-Perron et al (Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors, Gene, 33, p 103-119 (1985)) .
  • coding nucleotide sequence 5 comprising several (multiple) unique cloning sites (MCS) means a short coding sequence (or polylinker) comprising several cleavage sites for restriction enzymes.
  • nucleotide sequence 6 coding for a poison protein is understood to mean any (wild-type) nucleotide structure coding for a protein exhibiting naturally poison and specific activity on one or more vital functions of a host cell.
  • a poison protein is also characterized by the existence of an antidote or poison, such as the protein CcdB and CcdA, the protein Kid and its antagonist Kis, the protein Pe K and its antagonist PemI, the protein Doc and antagonist Phd, the HoK protein and its antagonist Sok and other poison molecules of plasmid origin or not.
  • an antidote or poison such as the protein CcdB and CcdA, the protein Kid and its antagonist Kis, the protein Pe K and its antagonist PemI, the protein Doc and antagonist Phd, the HoK protein and its antagonist Sok and other poison molecules of plasmid origin or not.
  • the nucleotide sequence 6 coding for a poison protein consists of the wild-type CcdB gene coding for the protein CcdB (Control of Cell Death) obtained from the ccd locus of the plasmid F (FIG. 2).
  • the ccd locus of plasmid F includes the two wild genes, ccdA and ccdB, also named H and G, or letA and letD, which code for proteins of 72 and 101 amino acids respectively (Bex et al, Mini-F encoded proteins; identification of a new 10.5 kilodalton species.
  • the protein CcdB of plasmid F is a cytotoxin whose lethal activity is antagonized by the protein CcdA (Karoui et al, Ham22, a mini-F mutation which is lethal to host cell and promoted recA-dependent induction of lambdoid prophage, EMBO J.2, 1863-1868 (1983); Ogura and Hiraga Mini-F plasmid gene that couple host cell division to plasmid proliferation, Proc. Natl. Acad. Sci. USA, 80, 4784-4788 (1983); Miki et al, Control of cell division by sex factor F in Escherichia coli. Identification of genes for inhibitor protein and trigger protein on the 42.84-43.6F segment, J. Mol. Biol. 174, 627-646 (1984b)).
  • the molecular mechanism by which the CcdB protein exerts its lethal activity has been elucidated; the protein CcdB is a poison of DNA-topoisomerase II.
  • Type II DNA topoisomerases are essential and ubiquitous enzymes that alter the topology of DNA by temporarily introducing a double-strand break in DNA.
  • topoisomerase II forms an intermediate complex with its substrate DNA in which the enzyme is covalently attached to the 5 'end of the cleaved DNA.
  • This transient intermediate in which topoisomerase II is covalently linked to DNA has been called the "cleavable complex" - (Wang, DNA topoisomerases. Annu. Rev. Biochem.
  • the cleavable topoisomerase II-DNA complex is the target of powerful therapeutic agents, including antibiotics of the "quinolone" family, which act on gyrase (bacterial topoisomerase II) and agents anticancer drugs (acridines, epipodophylotoxins), which act on topoisomerase II in mammals.
  • antibiotics of the "quinolone” family which act on gyrase (bacterial topoisomerase II) and agents anticancer drugs (acridines, epipodophylotoxins), which act on topoisomerase II in mammals.
  • acridines, epipodophylotoxins agents that act on topoisomerase II in mammals.
  • the therapeutic efficacy of topoisomerase poisons is correlated with their ability to stabilize the cleavable complex.
  • DNA topoisomerase II is an essential enzyme in all living things and very conserved in the evolution of species.
  • the protein CcdB therefore exhibits potential cytotoxic activity among a wide variety of prokaryotic species.
  • the small size of the wild ccdB gene allows inserting it into plasmids without excessively increasing its size and consequently makes it possible to include large fragments of foreign DNA therein.
  • the wild-type ccdB gene of plasmid F contains very few restriction sites, it is therefore easier to preserve the uniqueness of the multiple cloning sites (MCS) added to it.
  • the inventors have found that the phase fusion of the nucleotide sequence 6 coding for the protein CcdB with the coding nucleotide sequence (polylinker 5) comprising several (multiple) unique cloning sites (MCS), gives a nucleotide sequence 4 coding for a fusion protein active as a poison which consequently makes it possible to produce vectors for the direct selection of the recombinant plasmids ("killer selection").
  • the plasmids obtained make it possible to clone the restriction fragments doubly digested in the two orientations with respect to the lac promoter.
  • the insertion of a restriction fragment into one of the unique cloning sites interrupts the genetic information of the fusion gene which leads to the synthesis of a non-functional fusion gene product. Insertional inactivation of the fusion gene should always take place when a termination codon is introduced or when a reading phase change is created.
  • Cells harboring such an intact cloning vector produce a functional fusion poison protein and die.
  • the ccdB gene was amplified by PCR using as plasmid DNA, the plasmid pULB2208 (Bernard and Couturier, The 41 carboxy-terminal residues of the iniF plasmid CcdA protein are sufficient to antagonize the killer activity of the CcdB protein, Mol. Gen. Genet 226, 297-304 (1991)) as well as synthetic oligonucleotides.
  • the sequences of the synthetic oligonucleotides were chosen so as to create an EcoRI restriction site on either side of the wild-type ccdB gene in order to be able to reclonate this gene in phase with the codons of the multiple cloning sites MCS19 and to eliminate the codon initiation of the native ccdB gene.
  • the DNA resulting from the PCR reaction was digested with the EcoRI enzyme and cloned into the EcoRI site of the plasmid pUC19.
  • the plasmid pKIL2 is lethal for a wild bacterium (sensitive CcdB s ).
  • PKIL2 still has two SmaI sites, one at the multiple cloning sites, the other in the central region of the ccdB gene. The latter was eliminated by site-specific multagenesis.
  • the resulting plasmid, pKIL19 (FIG. 3) therefore has a unique EcoRI restriction site at the level of the sequence 5 comprising the multiple cloning sites.
  • Example II construction of the plasmid PKIL18
  • the ccdB gene was amplified by PCR using as DNA template, the plasmid pKIL19 as well as synthetic oligonucleotides.
  • the sequences of the synthetic oligonucleotides were chosen so as to create a HindIII site on either side of the ccdB gene in order to be able to reclonate this gene in phase with the codons of the multiple MCS18 cloning sites.
  • DNA from the reaction of PCR was digested with the enzyme HindIII and cloned into the HindIII site of the plasmid pUCIS.
  • the resulting plasmid having integrated the HindIII fragment in the orientation allowing the reading, from the Lac promoter, of the ccdB gene endowed with additional codons corresponding to the multiple cloning sites MCS18, was called pKIL4.
  • the plasmid pKIL4 is lethal for a sensitive bacterium CcdB s .
  • the latter must be resistant to the lethal effect of the active fusion protein as a poison.
  • the chromosomal mutation gyrA462 gives the strains total resistance to the poison effect of the fusion protein.
  • the plasmids pKIL18 and pKIL19 deriving directly from the plasmids pUC18 and pUC19 and expressing the ccdB genes from the Lac promoter it is preferable to maintain these plasmids in a LacJ q strain.
  • strain acJ q makes it possible to reduce the expression from the Lac promoter and saves the bacterial machinery which makes it possible to guarantee a rapid generation time (high production of the vector by the strain).
  • the strain D1210 (Sadler et al Gene 8, p 279-300 (1980)) derived from the strain HB101 Lacl ⁇ , LacY * (Maniatis et al (Molecular Cloning Laboratories Man. Cold Spring Harbor Laboratory NY), characterized by an I q chromosome and a high transformation rate, was transformed by the plasmid pC0S2.1 This plasmid, which confers resistance to kanamycin, carries the recA gene of Erwinia chrysanthemi 3665, and allows the recombination in E. coli.
  • phage PI was prepared on a strain CcdB R gyrA462, zei- 298 :: TnlO and used to infect the strain D1210 / pCOS2.1. Tetracycline resistant transductants were selected and tested for their resistance or sensitivity to the CcdB protein. One of the CcdB R transductants was then cleared from the plasmid pCOS2.1 and named KIB22.
  • the strain KIB22 constitutes an ideal host strain for the plasmids pKIL18 and pKIL19 while the strain D1210 constitutes the ideal host for the selection of the recombinant plasmids.
  • the strain KIB22 advantageously has a high DNA extraction rate (comparable to the yield of plasmids pUC) and unexpectedly resistance to the fusion protein encoded by pKILl ⁇ and pKIL19.
  • the cloning vector according to the invention in numerous copies without causing the death of said microorganism.
  • the selection is made by simple spreading of the bacteria on a medium containing IPTG (Isopropyl-Beta-D-thiogalacto-pyranoside), as well as ampicillin.
  • the strain KIB22 has been deposited with the Laboratorium voor Microbiologie-Bacteri ⁇ nverzameling (LMG) of the Belgian Coordinated Collections of Microorganisms (BCCM) under the number LMG P-12601.
  • LMG Laboratorium voor Microbiologie-Bacteri ⁇ nverzameling
  • BCCM Belgian Coordinated Collections of Microorganisms
  • the cloning vector pKIL19 has been deposited with the Laboratorium voor Molé Diagram Biologie-Plasmiden Collectie (LMBP) of the Belgian Coordinated Collections of Microorganisms (BCCM) under the number LMBP 2781.
  • LMBP Laboratorium voor Molé Diagram Biologie-Plasmiden Collectie
  • BCCM Belgian Coordinated Collections of Microorganisms

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Abstract

A cloning and/or sequencing vector (1) comprises, incorporated in an autonomous replication vector (2), at least one nucleotide promoter sequence (3) and at least one nucleotide sequence (4) coding for a fusion protein active as a poison. Said nucleotide sequence (4) is obtained by fusion of a coding nucleotide sequence (5), comprising several unique cloning sites and a nucleotide sequence (6) coding for a poison protein. The vector host cell of the invention is also disclosed.

Description

VECTEUR DE CLONAGE ET/OU DE SEOUENCAGE Objet de l'invention OBJECT OF THE INVENTION CLONING AND / OR SEQUENCING VECTOR
L'invention concerne un vecteur de clonage et/ou de séquençage permettant une sélection directe de clones recombinants.The invention relates to a cloning and / or sequencing vector allowing direct selection of recombinant clones.
L'invention concerne également la cellule procaryote transformée par ce vecteur et la cellule procaryote hôte de ce vecteur ainsi que l'utilisation de ce vecteur de clonage et de séquençage pour la sélection et le séquençage de clones recombinants.The invention also relates to the prokaryotic cell transformed by this vector and the prokaryotic host cell of this vector as well as to the use of this cloning and sequencing vector for the selection and sequencing of recombinant clones.
Etat de la technique et arrière plan technologique à la base de l'inventionState of the art and technological background underlying the invention
Des vecteurs de clonage phagiques (série des M13) et plasmidiques (série des pUC) contenant de nombreux sites uniques de clonage ont été construits par Messing et al (P.N.A.S. USA, 79, p 3642-3646 (1977), par Norrander et al (Gène, 26, p 101-106 (1983) et Yanisch-perron et al (Gène, 33 p 103 à 119 (1985) ) .Phage (M13 series) and plasmid (pUC series) cloning vectors containing many unique cloning sites were constructed by Messing et al (PNAS USA, 79, p 3642-3646 (1977), by Norrander et al ( Gene, 26, p 101-106 (1983) and Yanisch-perron et al (Gene, 33 p 103-119 (1985)).
Les multiples sites de clonage (MCS - multiples cloning sites) de ces vecteurs sont localisés dans la séquence codante du gène LacZ. La discrimination entre les cellules transformées hébergeant un vecteur recombinant et les cellules hébergeant un vecteur non recombinant, s'effectue par la technique duThe multiple cloning sites (MCS - multiple cloning sites) of these vectors are located in the coding sequence of the LacZ gene. The discrimination between the transformed cells harboring a recombinant vector and the cells harboring a non-recombinant vector, is carried out by the technique of
"Blue Screen" décrite par Gronenborn et Messing (Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavages sites, Nature, 272, p 375-377 (1978)) ."Blue Screen" described by Gronenborn and Messing (Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavages sites, Nature, 272, p 375-377 (1978)).
Cependant, cette technique du "Blue Screen" présente l'inconvénient d'utiliser un procédé de criblageHowever, this "Blue Screen" technique has the disadvantage of using a screening process
(descrimination) et non un procédé de sélection des clones.(descrimination) and not a process for selecting clones.
La discrimination par criblage est basée sur l'identification d'un clone au sein d'une population de clones sur base d'une caractéristique (couleur) qui le différencie. La sélection ne nécessite pas cette caractéristique, puisque par cette méthode, on isole uniquement les clones recombinants.Screening discrimination is based on the identification of a clone within a population of clones on the basis of a characteristic (color) which differentiates it. The selection does not require this characteristic, since by this method, we isolate only recombinant clones.
Le procédé de criblage est basé sur la coloration des clones recombinants (coloration blanche) et des clones non recombinants (coloration bleue) . Cette coloration est basée sur 1 ' inactivation du marqueur Beta-galactosidase, prévenant le clivage de 1 'X-gal (5-bromo-4-chloro-3-indolyl- b-galactoside) . Les colonies cellulaires hébergeant un vecteur non recombinant produisent une Beta-galactosidase fonctionnelle et en hydrolysant le substrat X-gal, produisent un colorant bleu. L'insertion d'un fragment d'ADN dans le gène de la Beta-galactosidase, prévient généralement le clivage de l'X-gal. Les cellules hébergeant un vecteur recombinant ont donc une coloration blanche.The screening process is based on the coloration of the recombinant clones (white coloration) and of the non-recombinant clones (blue coloration). This staining is based on the inactivation of the beta-galactosidase marker, preventing cleavage of the X-gal (5-bromo-4-chloro-3-indolyl-b-galactoside). Cell colonies harboring a non-recombinant vector produce a functional beta-galactosidase and by hydrolyzing the X-gal substrate produce a blue dye. The insertion of a DNA fragment into the Beta-galactosidase gene, generally prevents the cleavage of X-gal. The cells hosting a recombinant vector therefore have a white coloration.
En outre, ce procédé complexe nécessite l'utilisation du substrat X-gal qui est un produit fort coûteux, instable et d'utilisation délicate.In addition, this complex process requires the use of the X-gal substrate which is a very expensive product, unstable and delicate to use.
D'autre part, divers vecteurs de clonage permettant de sélectionner directement (sélection positive) des souches recombinantes, ont été décrits dans la littérature scientifique.On the other hand, various cloning vectors allowing direct selection (positive selection) of the recombinant strains have been described in the scientific literature.
Pierce et al (Proc. Natl. Acad. Sci., vol 89, n°6Pierce et al (Proc. Natl. Acad. Sci., Vol 89, n ° 6
1992, p 2056-2060) décrivent un vecteur comprenant le gène létal sac B de Bacillus amyloliquefaciens, intégré dans un plasmide dérivé du bactériophage PI, sous le contrôle d'un promoteur spécifique de E. coli.1992, p 2056-2060) describe a vector comprising the lethal sac B gene of Bacillus amyloliquefaciens, integrated into a plasmid derived from bacteriophage PI, under the control of a promoter specific for E. coli.
Le promoteur de ce vecteur comporte une région avec plusieurs sites spécifiques de clonage (site de clivage pour une enzyme de restriction) .The promoter of this vector comprises a region with several specific cloning sites (cleavage site for a restriction enzyme).
Comme le gène sac B code pour le levansucrase catalysant l'hydrolyse du sucrose en produits toxiques pour E. coli; la sélection directe des mutants intégrant un plasmide recombiné, se fait sur un milieu de culture contenant du sucrose. Comme le levansucrase est toxique, même en l'absence de sucrose, il est donc indispensable de réprimer sa synthèse si l'on désire obtenir un grand nombre de copies plasmidiques dans le cytoplasme bactérien.As the sac B gene codes for levansucrase catalyzing the hydrolysis of sucrose into products toxic for E. coli; the direct selection of the mutants integrating a recombinant plasmid is done on a culture medium containing sucrose. As levansucrase is toxic, even in the absence of sucrose, it is therefore essential to suppress its synthesis if one wishes to obtain a large number of plasmid copies in the bacterial cytoplasm.
Cependant une répression totale du gène cytotoxique est difficile, voire impossible; particulièrement si on désire avoir un grand nombre de copies du vecteur.However, total repression of the cytotoxic gene is difficult, if not impossible; especially if we want to have a large number of copies of the vector.
Aussi, l'impossibilité de réprimer le gène cytotoxique entraîne dans les phases de production du plasmide, la mort de la cellule et par conséquent une pression sélective vers des souches mutées (caractérisées par un gène létal inactif) .Also, the impossibility of repressing the cytotoxic gene leads in the plasmid production phases, cell death and therefore a selective pressure towards mutated strains (characterized by an inactive lethal gene).
Dans cas ci, pour que l'enzyme codée par le gène sac B ne tue pas la cellule hôte, il est nécessaire d'incorporé au vecteur de clonage un répresseur CI qui régule l'expression de ce gène.In this case, so that the enzyme coded by the sac B gene does not kill the host cell, it is necessary to incorporate into the cloning vector a CI repressor which regulates the expression of this gene.
De plus, comme le sucrose est souvent incorporé au milieux de culture bactériens, il sera indispensable de préparer des milieux totalement exempt de sucrose pour pratiquer ces manipulations. Henrich et al (Gène, vol 42, n°3, 1986, p 345-349) décrivent un vecteur comprenant le gène E issu du bactériophage ψ XllΛ , ledit gène E étant incorporé dans le plasmide pUH84, sous le contrôle du promoteur Lac.In addition, as sucrose is often incorporated into bacterial culture media, it will be essential to prepare media completely free of sucrose to practice these manipulations. Henrich et al (Gene, vol 42, n ° 3, 1986, p 345-349) describe a vector comprising the E gene derived from the bacteriophage ψ XllΛ, said E gene being incorporated into the plasmid pUH84, under the control of the Lac promoter.
Dans ce cas, le gène E comporte six sites uniques de restriction (localisés sur toute la séquence du gène E) et code pour la gpE qui provoque la lyse cellulaire de E. coli. Dans ce cas, la sélection positive s'effectue lorsque un gène étranger recombinant est venu s ' insérer au niveau d'un des sites de restriction. Cependant, cette insertion d'un gène étranger au niveau d'un site de restriction localisé dans la séquence du gène E, codant pour la gpE, rend plus difficile le séquençage et/ou l'amplification par PCR du gène étranger, car dans ce cas on séquence, on amplifie et on caractérise également des portions de séquences inutiles appartenant au gène E codant pour la gpE.In this case, the E gene has six unique restriction sites (located throughout the E gene sequence) and codes for the gpE which causes cell lysis of E. coli. In this case, the positive selection is carried out when a recombinant foreign gene has come to insert itself at one of the restriction sites. However, this insertion of a foreign gene at a restriction site located in the sequence of the E gene, coding for gpE, makes sequencing and / or PCR amplification of the foreign gene more difficult, because in this if it is sequenced, amplified and also characterized portions of unnecessary sequences belonging to the E gene coding for gpE.
Kuhn et al (Gène, vol 42, n°3, 1986, p 253-263) décrivent un vecteur comprenant un gène de grande taille, codant pour une enzyme de restriction qui tue par clivage du génome de la bactérie, ledit gène étant incorporé dans le plasmide pKG2 sous le contrôle du promoteur LacUV5.Kuhn et al (Gene, vol 42, n ° 3, 1986, p 253-263) describe a vector comprising a large gene, coding for a restriction enzyme which kills by cleavage of the genome of the bacterium, said gene being incorporated in the plasmid pKG2 under the control of the LacUV5 promoter.
Les vecteurs de clonage de l'état de la technique présentent l'inconvénient de devoir être maintenu dans une souche hôte, comportant le represseur Laclq sous forme épisomique ou le represseur CI, afin d1 inactiver le promoteur d'empêcher l'expression du gène tueur et d'entraîner ainsi la mort de la souche hôte. Aussi, si l'on désire faire produire par cette souche un grand nombre de copies des vecteurs de clonage, le represseur, ne sera pas suffisant pour empêcher soit une pression sélective modifiant l'activité cytotoxique du vecteur, soit une "fuite génétique", c'est-à-dire l'expression de certaines copies du vecteur et la mort de la souche hôte.Cloning vectors of the state of the art have the disadvantage of having to be maintained in a host strain, containing the LacI repressor q episomally or repressor CI, to one inactivate the promoter prevent the expression of the killer gene and thereby cause the death of the host strain. Also, if it is desired to cause this strain to produce a large number of copies of the cloning vectors, the repressor will not be sufficient to prevent either a selective pressure modifying the cytotoxic activity of the vector, or a "genetic leakage", that is, the expression of certain copies of the vector and the death of the host strain.
Par conséquent, aucun des documents de l'état de la technique ne décrit un vecteur de clonage pouvant incorporer des fragments nucléotidiques de grande taille, qui soit de manipulation aisée et que puisse être produit de manière industrielle par un microorganisme; c'est-à-dire qu'il puisse être produit en grand nombre de copies par un microorganisme sans occasionner la mort de celui-ci. Buts de l'invention La présente invention vise à fournir un nouveau vecteur de clonage et/ou de séquençage, ainsi que sa souche hôte, qui soient de construction et de production aisées et peu coûteuses, et qui permettent la sélection directe de clones recombinants, sans présenter les inconvénients de l'état de la technique susmentionné.Consequently, none of the documents of the prior art describes a cloning vector which can incorporate large nucleotide fragments, which is easy to handle and which can be produced industrially by a microorganism; that is, it can be produced in large numbers of copies by a microorganism without causing its death. AIMS OF THE INVENTION The present invention aims to provide a new cloning and / or sequencing vector, as well as its host strain, which are of easy and inexpensive construction and production, and which allow the direct selection of recombinant clones, without having the drawbacks of the aforementioned prior art.
Un but particulier de la présente invention est d'obtenir un vecteur qui permet une sélection spécifique et certaine des clones recombinants.A particular object of the present invention is to obtain a vector which allows specific and certain selection of the recombinant clones.
Un autre but de la présente invention vise à obtenir un vecteur qui permet le séquençage, l'amplification et/ou la caractérisation avec la même amorce, de n'importe quel fragment d'ADN étranger (quelque soit sa taille) dans des clones recombinants.Another object of the present invention is to obtain a vector which allows the sequencing, amplification and / or characterization with the same primer, of any foreign DNA fragment (whatever its size) in recombinant clones .
Un but supplémentaire de la présente invention, vise à obtenir un vecteur qui permet également une extraction aisée de ce fragment d'ADN étranger, du clone recombinant.Another object of the present invention is to obtain a vector which also allows easy extraction of this fragment of foreign DNA from the recombinant clone.
Un dernier but de la présente invention vise à obtenir une souche hôte dudit vecteur qui permette d'obtenir la production d'un grand nombre de copies dudit vecteur sans occasionner de pression sélection modifiant l'activité cytotoxique dudit vecteur ou la mort de la souche hôte. Eléments caractéristiques de l'invention L'invention concerne un nouveau vecteur de clonage et/ou de séquençage, comprenant, incorporé dans un vecteur à réplication autonome, au moins une séquence nucléotidique promotrice et au moins une séquence nucléotidique codant pour une protéine de fusion active en tant que poison; ladite séquence nucléotidique étant obtenue par la fusion d'une séquence nucléotidique codante comprenant plusieurs sites uniques de clonage et d'une séquence nucléotidique codant pour une protéine poison.A final object of the present invention aims to obtain a host strain of said vector which makes it possible to obtain the production of a large number of copies of said vector without causing selection pressure modifying the cytotoxic activity of said vector or the death of the host strain. Characteristic Elements of the Invention The invention relates to a new cloning and / or sequencing vector, comprising, incorporated into an autonomous replicating vector, at least one promoter nucleotide sequence and at least one nucleotide sequence coding for an active fusion protein as poison; said nucleotide sequence being obtained by the fusion of a coding nucleotide sequence comprising several unique cloning sites and of a nucleotide sequence coding for a poison protein.
De préférence, le vecteur à réplication autonome est un virus ou un plasmide tel qu'un plasmide pUC, recombinant.Preferably, the autonomously replicating vector is a virus or a plasmid such as a recombinant pUC plasmid.
La séquence nucléotidique promotrice, peut comprendre n'importe quel promoteur, permettant 1'expression de la séquence nucléotidique codant pour une protéine de fusion active en tant que poison.The promoter nucleotide sequence can include any promoter, allowing expression of the nucleotide sequence encoding an active fusion protein as a poison.
De préférence, cette séquence nucléotidique promotrice est constituée par le promoteur de l'opéron Lac. Selon une forme d'exécution préférée de l'invention, les sites uniques de clonage (MCS) de la séquence nucléotidique fusionnée à la séquence nucléotidique codant pour la protéine poison sont absents du reste de la séquence nucléotidique du vecteur selon l'invention.Preferably, this promoter nucleotide sequence is constituted by the promoter of the Lac operon. According to a preferred embodiment of the invention, the unique cloning sites (MCS) of the nucleotide sequence fused to the nucleotide sequence coding for the poison protein are absent from the rest of the nucleotide sequence of the vector according to the invention.
Avantageusement, la séquence nucléotidique du gène codant pour la protéine poison comprend tout ou une partie de la séquence nucléotidique du gène sauvage codant pour la protéine CcdB.Advantageously, the nucleotide sequence of the gene coding for the poison protein comprises all or part of the nucleotide sequence of the wild-type gene coding for the protein CcdB.
De préférence, la séquence nucléotidique du gène codant pour la protéine poison est dépourvue du site de clivage de l'enzyme de restriction S al. Un autre aspect de l'invention, concerne une cellule procaryote transformée par le vecteur de clonage selon 1'invention.Preferably, the nucleotide sequence of the gene coding for the poison protein is devoid of the cleavage site of the restriction enzyme S al. Another aspect of the invention relates to a prokaryotic cell transformed with the cloning vector according to the invention.
L'invention concerne également une cellule procaryote hôte du vecteur selon l'invention, comportant un Iq chromosomique, un taux de transformation élevé et comportant une mutation conférant la résistance à l'activité poison de la protéine de fusion et/ou comportant un gène codant pour une protéine antipoison de la protéine de fusion. De préférence, la cellule procaryote hôte du vecteur selon l'invention, comporte une mutation dans le gène codant pour la sous unité A ou dans le gène codant pour la sous unité B de la gyrase et conférant la résistance à la protéine de fusion et/ou un gène codant pour la protéine CcdA antipoison de la protéine de fusion.The invention also relates to a cell. prokaryotic host of the vector according to the invention, comprising a chromosomal I q , a high transformation rate and comprising a mutation conferring resistance to the poison activity of the fusion protein and / or comprising a gene coding for a poison protein of the fusion protein. Preferably, the prokaryotic host cell of the vector according to the invention comprises a mutation in the gene coding for the subunit A or in the gene coding for the subunit B of the gyrase and conferring resistance to the fusion protein and / or a gene encoding the CcdA protein poison from the fusion protein.
Préférentiellement, la cellule procaryote est une cellule d'Eschierichia coli comportant une mutation responsable de la substitution de l'arginine 462 par une cysteine dans la séquence des acides aminés du polypeptide GyrA de la gyrase, conférant la résistance à la protéine de fusion.Preferably, the prokaryotic cell is an Eschierichia coli cell comprising a mutation responsible for the substitution of arginine 462 by a cysteine in the amino acid sequence of the gyrase GyrA polypeptide, conferring resistance to the fusion protein.
De préférence, cette cellule procaryote hôte comporte également la mutation Laclq . La présente invention concerne également des fragments du vecteur selon l'invention, en particulier des amorces de séquençages et/ou d'amplification (par exemple par PCR) des fragments nucléotidiques étrangers insérés dans le vecteur selon l'invention. De préférence, ces amorces sont constituées par des séquences de 10 à 30 nucléotides s'hybridant aux séquences nucléotidiques situées de part et d'autre de la séquence nucléotidique comprenant plusieurs sites uniques de clonage, du vecteur selon l'invention. Un dernier aspect de l'invention concerne l'utilisation du vecteur selon l'invention pour la sélection et le séquençage de clones recombinants. Brève description des figuresPreferably, this prokaryotic host cell also includes the Lacl q mutation. The present invention also relates to fragments of the vector according to the invention, in particular primers for sequencing and / or amplification (for example by PCR) of the foreign nucleotide fragments inserted into the vector according to the invention. Preferably, these primers consist of sequences of 10 to 30 nucleotides which hybridize to the nucleotide sequences located on either side of the nucleotide sequence comprising several unique cloning sites, of the vector according to the invention. A final aspect of the invention relates to the use of the vector according to the invention for the selection and sequencing of recombinant clones. Brief description of the figures
- La figure 1 représente de manière schématique un vecteur de clonage selon la présente invention.- Figure 1 schematically shows a cloning vector according to the present invention.
- La figure 2 représente la séquence nucléotidique du gène ccdB codant pour la protéine CcdB.- Figure 2 shows the nucleotide sequence of the ccdB gene coding for the protein CcdB.
- Les figures 3 et 4 représentent la séquence nucléotidique codant pour la protéine de fusion des vecteurs de clonages pKIL18 et pKIL19 respectivement. Ces séquences sont pourvues d'une séquence nucléotidique contenant de multiples sites uniques de clivage pour différents enzymes de restriction. Ces vecteurs de clonages pKIL18 et pKIL19 ont été obtenus par une recombinaison in vitro entre le gène sauvage ccdB du plasmide F et les plasmides pUC18 et pUC19 respectivement. Description d'une forme d'exécution préférée de l'invention Selon l'invention, le vecteur de clonage et/ou de séquençage 1 comprend incorporé dans un vecteur à réplication autonome 2, au moins une séquence nucléotidique promotrice 3 et au moins une séquence nucléotidique 4 codant pour une protéine de fusion active en tant que poison; ladite séquence nucléotidique 4 étant obtenue par la fusion d'une séquence nucléotidique codante 5 (ou polylinker) comprenant plusieurs (multiples) sites uniques de clonage (MCS) et d'une séquence nucléotidique 6 codant pour une protéine poison.- Figures 3 and 4 show the sequence nucleotide encoding the fusion protein of the cloning vectors pKIL18 and pKIL19 respectively. These sequences are provided with a nucleotide sequence containing multiple unique cleavage sites for different restriction enzymes. These pKIL18 and pKIL19 cloning vectors were obtained by in vitro recombination between the wild-type ccdB gene of plasmid F and the plasmids pUC18 and pUC19 respectively. Description of a preferred embodiment of the invention According to the invention, the cloning and / or sequencing vector 1 comprises incorporated into a self-replicating vector 2, at least one promoter nucleotide sequence 3 and at least one sequence nucleotide 4 encoding an active fusion protein as a poison; said nucleotide sequence 4 being obtained by the fusion of a coding nucleotide sequence 5 (or polylinker) comprising several (multiple) unique cloning sites (MCS) and of a nucleotide sequence 6 coding for a poison protein.
On entend par vecteur à réplication autonome 2, toute construction nucléotidique tel qu'un virus ou un plasmide (de préférence un plasmide de la série PUC recombinant) susceptible de s'introduire dans un microorganisme, de s'y recombiner et/ou de s'y répliquer.The term “autonomously replicating vector 2” is intended to mean any nucleotide construct such as a virus or a plasmid (preferably a recombinant PUC series plasmid) capable of entering a microorganism, of recombining therein and / or of s 'replicate it.
La figure 1, représente de manière schématique un vecteur de clonage selon la présente invention, construit à partir d'un plasmide de la série pUC (pUC18 et pUC19) décrite par Norrander et al (Construction of improved M13 vectors using oligodeoxinucleotide-directed mutagenesis, Gène, 26, p 101-106 (1983)) et par Yanisch-Perron et al (Improved M13 phage cloning vectors and host strains : nucleotide séquences of the M13mpl8 and pUC19 vectors, Gène, 33, p 103-119 (1985)) .FIG. 1 schematically represents a cloning vector according to the present invention, constructed from a plasmid of the pUC series (pUC18 and pUC19) described by Norrander et al (Construction of improved M13 vectors using oligodeoxinucleotide-directed mutagenesis, Gene, 26, p 101-106 (1983)) and by Yanisch-Perron et al (Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors, Gene, 33, p 103-119 (1985)) .
On entend par séquence nucléotidique codante 5 comprenant plusieurs (multiples) sites uniques de clonage (MCS) une courte séquence codante (ou polylinker) comprenant plusieurs sites de clivage pour des enzymes de restriction.The term coding nucleotide sequence 5 comprising several (multiple) unique cloning sites (MCS) means a short coding sequence (or polylinker) comprising several cleavage sites for restriction enzymes.
L'avantage d'un polylinker dans le vecteur selon l'invention est la localisation sur une seule courte séquence de différents sites de clonage qui permet :The advantage of a polylinker in the vector according to the invention is the localization on a single short sequence different cloning sites which allow:
- la séquence et l'amplification rapide avec les mêmes amorces, de n'importe quel fragment d'ADN inséré dans ce vecteur. - l'extraction rapide de fragment clone, facilité par la proximité des sites de restriction. En effet, contrairement à l'état de la technique cette proximité évite de séquencer, d'amplifier et de caractériser des fragments inutiles des autres séquences du vecteur selon l'invention. On entend par séquence nucléotidique 6 codant pour une protéine poison, toute structure nucléotidique (sauvage) codant pour une protéine présentant une activité naturellement poison et spécifique sur une ou plusieurs fonctions vitales d'une cellule hôte. Une protéine poison est également caractérisée par l'existence d'un antidote ou antipoison, tel que la protéine CcdB et CcdA, la protéine Kid et son antagoniste Kis, la protéine Pe K et son antagoniste PemI, la protéine Doc et antagoniste Phd, la protéine HoK et son antogiste Sok et d'autres molécules poisons d'origine plasmidiques ou non.- the sequence and rapid amplification with the same primers, of any DNA fragment inserted into this vector. - rapid extraction of the cloned fragment, facilitated by the proximity of the restriction sites. In fact, unlike the prior art, this proximity avoids sequencing, amplifying and characterizing unnecessary fragments of the other sequences of the vector according to the invention. The term nucleotide sequence 6 coding for a poison protein is understood to mean any (wild-type) nucleotide structure coding for a protein exhibiting naturally poison and specific activity on one or more vital functions of a host cell. A poison protein is also characterized by the existence of an antidote or poison, such as the protein CcdB and CcdA, the protein Kid and its antagonist Kis, the protein Pe K and its antagonist PemI, the protein Doc and antagonist Phd, the HoK protein and its antagonist Sok and other poison molecules of plasmid origin or not.
Dans ce cas, la séquence nucléotidique 6 codant pour une protéine poison est constituée par le gène sauvage CcdB codant pour la protéine CcdB (Control of Cell Death) obtenue à partir du locus ccd du plasmide F (figure 2) . Le locus ccd du plasmide F, comprend les deux gènes sauvages, ccdA et ccdB, également nommés H et G, ou letA et letD, et qui codent respectivement pour des protéines de 72 et 101 acides aminés (Bex et al, Mini-F encoded proteins; identification of a new 10.5 kilodalton species. EMBO J.2, 1853-1861 (1983) ; Miki et al, Control of cell division by sex factor F in Escherichia coli. I. The 42.84-43.6 F segment couples cell division of the host bacteria with réplication of plasmid DNA, J. Mol. Bio. , 174, 605-625, (1984) ) .In this case, the nucleotide sequence 6 coding for a poison protein consists of the wild-type CcdB gene coding for the protein CcdB (Control of Cell Death) obtained from the ccd locus of the plasmid F (FIG. 2). The ccd locus of plasmid F, includes the two wild genes, ccdA and ccdB, also named H and G, or letA and letD, which code for proteins of 72 and 101 amino acids respectively (Bex et al, Mini-F encoded proteins; identification of a new 10.5 kilodalton species. EMBO J.2, 1853-1861 (1983); Miki et al, Control of cell division by sex factor F in Escherichia coli. I. The 42.84-43.6 F segment couples cell division of the host bacteria with replication of plasmid DNA, J. Mol. Bio., 174, 605-625, (1984)).
Chez Escherichia coli, la protéine CcdB du plasmide F est une cytotoxine dont l'activité létale est antagonisée par la protéine CcdA (Karoui et al, Ham22, a mini-F mutation which is lethal to host cell and promûtes recA-dependent induction of lambdoid prophage. EMBO J.2, 1863-1868 (1983) ; Ogura and Hiraga Mini-F plasmid gène that couple host cell division to plasmid prolifération, Proc. Natl. Acad. Sci. USA, 80, 4784-4788 (1983) ; Miki et al, Control of cell division by sex factor F in Escherichia coli. Identification of gènes for inhibitor protein and trigger protein on the 42.84-43.6F segment, J. Mol. Biol. 174, 627-646 (1984b)) .In Escherichia coli, the protein CcdB of plasmid F is a cytotoxin whose lethal activity is antagonized by the protein CcdA (Karoui et al, Ham22, a mini-F mutation which is lethal to host cell and promoted recA-dependent induction of lambdoid prophage, EMBO J.2, 1863-1868 (1983); Ogura and Hiraga Mini-F plasmid gene that couple host cell division to plasmid proliferation, Proc. Natl. Acad. Sci. USA, 80, 4784-4788 (1983); Miki et al, Control of cell division by sex factor F in Escherichia coli. Identification of genes for inhibitor protein and trigger protein on the 42.84-43.6F segment, J. Mol. Biol. 174, 627-646 (1984b)).
Le mécanisme moléculaire par lequel la protéine CcdB exerce son activité létale a été élucidé ; la protéine CcdB est un poison de la DNA-topoisomérase II. Les DNA topoisomérases de type II sont des enzymes essentiels et ubiquiεtes qui altèrent la topologie du DNA en introduisant transitoirement une cassure double-brin dans le DNA. Durant l'étape de cassure-religation, la topoisomerase II forme un complexe intermédiaire avec son DNA substrat dans lequelle l'enzyme est attaché de manière covalente à l'extrémité 5' du DNA clivé. Cet intermédiaire transitoire dans lequel la topoisomerase II est liée de manière covalente au DNA a été nommé le "complexe clivable"— (Wang, DNA topoisomérases. Annu. Rev. Biochem. 54, 665-97, 1985; Maxwell S. Gellert, Mechanistic aspects of DNA topoisomérases. Advan. Protein Che . 38, 69-107, 1986; Liu, DNA topoisomerase poisons as antitumor drugs, Annu. Rev. Biochem. 58. 351-375, 1989) .The molecular mechanism by which the CcdB protein exerts its lethal activity has been elucidated; the protein CcdB is a poison of DNA-topoisomerase II. Type II DNA topoisomerases are essential and ubiquitous enzymes that alter the topology of DNA by temporarily introducing a double-strand break in DNA. During the break-religation step, topoisomerase II forms an intermediate complex with its substrate DNA in which the enzyme is covalently attached to the 5 'end of the cleaved DNA. This transient intermediate in which topoisomerase II is covalently linked to DNA has been called the "cleavable complex" - (Wang, DNA topoisomerases. Annu. Rev. Biochem. 54, 665-97, 1985; Maxwell S. Gellert, Mechanistic aspects of DNA topoisomerases. Advan. Protein Che. 38, 69-107, 1986; Liu, DNA topoisomerase poisons as antitumor drugs, Annu. Rev. Biochem. 58. 351-375, 1989).
Aussi bien chez les eucaryotes que chez les procaryotes, le complexe clivable topoisomerase II-DNA, est la cible de puissants agents thérapeutiques, incluant les antibiotiques de la famille des "quinolones", qui agissent sur la gyrase (topoisomerase II bactérienne) et des agents anticancéreux (acridines, epipodophylotoxins) , qui agissent sur la topoisomerase II des mammifères. L'efficacité thérapeutique des poisons des topoisomérases est corrélée à leur capacité à stabiliser le complexe clivable.In both eukaryotes and prokaryotes, the cleavable topoisomerase II-DNA complex is the target of powerful therapeutic agents, including antibiotics of the "quinolone" family, which act on gyrase (bacterial topoisomerase II) and agents anticancer drugs (acridines, epipodophylotoxins), which act on topoisomerase II in mammals. The therapeutic efficacy of topoisomerase poisons is correlated with their ability to stabilize the cleavable complex.
La DNA-topoisomérase II est une enzyme essentielle chez tous les êtres vivants et très conservée dans l'évolution des espèces. La protéine CcdB présente donc une activité cytotoxique potentielle parmi une grande variété d'espèces procaryotiques.DNA topoisomerase II is an essential enzyme in all living things and very conserved in the evolution of species. The protein CcdB therefore exhibits potential cytotoxic activity among a wide variety of prokaryotic species.
La petite taille du gène sauvage ccdB permet de l'insérer dans des plasmides sans en augmenter exagérément la taille et permet par conséquent d'y inclure des fragments d'ADN étrangers de grandes tailles. De plus, étant donné sa petite taille, le gène sauvage ccdB du plasmide F contient très peu de sites de restriction, il est donc plus aisé de préserver l'unicité des sites multiples de clonage (MCS) qui lui sont ajoutés.The small size of the wild ccdB gene allows inserting it into plasmids without excessively increasing its size and consequently makes it possible to include large fragments of foreign DNA therein. In addition, given its small size, the wild-type ccdB gene of plasmid F contains very few restriction sites, it is therefore easier to preserve the uniqueness of the multiple cloning sites (MCS) added to it.
De manière inattendue, les inventeurs ont constaté que la fusion en phase de la séquence nucléotidique 6 codant pour la protéine CcdB avec la séquence nucléotidique codante (polylinker 5) comprenant plusieurs (multiples) sites uniques de clonage (MCS) , donnait une séquence nucléotidique 4 codant pour une protéine de fusion active en tant que poison qui permet par conséquent de produire des vecteurs de sélection ' directe des plasmides recombinants ("killer sélection") .Unexpectedly, the inventors have found that the phase fusion of the nucleotide sequence 6 coding for the protein CcdB with the coding nucleotide sequence (polylinker 5) comprising several (multiple) unique cloning sites (MCS), gives a nucleotide sequence 4 coding for a fusion protein active as a poison which consequently makes it possible to produce vectors for the direct selection of the recombinant plasmids ("killer selection").
Les plasmides obtenus permettent de cloner les fragments de restriction doublement digérés dans les deux orientations par rapport au promoteur lac. L'insertion d'un fragment de restriction dans un des sites uniques de clonage, interrompt l'information génétique du gène de fusion ce qui mène à la synthèse d'un produit de gène de fusion non fonctionnel. L' inactivation insertionelle du gène de fusion devrait toujours avoir lieu lorsqu'un codon de terminaison est introduit ou lorsqu'un changement de phase lecture est créé.The plasmids obtained make it possible to clone the restriction fragments doubly digested in the two orientations with respect to the lac promoter. The insertion of a restriction fragment into one of the unique cloning sites interrupts the genetic information of the fusion gene which leads to the synthesis of a non-functional fusion gene product. Insertional inactivation of the fusion gene should always take place when a termination codon is introduced or when a reading phase change is created.
Les cellules hébergeant un tel vecteur de clonage intact, produisent une protéine poison de fusion fon c- tionelle et meurent.Cells harboring such an intact cloning vector produce a functional fusion poison protein and die.
L'insertion d'un fragment d'ADN étranger dans un des sites uniques de clonage du gène de fusion, interfère avec la production du poison.The insertion of a foreign DNA fragment into one of the unique fusion gene cloning sites interferes with the production of the poison.
Les cellules hébergeant un vecteur recombinant seront viables, alors que celles hébergeant un vecteur intact seront non viables. Cette "Killer Sélection" par simple culture sur milieu solide permet d'éliminer les cellules hébergeant un vecteur non recombinant (clones non viables) et de sélectionner les clones recombinants (clones viables) . Exemple I ; Construction du plasmide PKIL19Cells harboring a recombinant vector will be viable, while cells harboring an intact vector will be non-viable. This "Killer Selection" by simple culture on solid medium makes it possible to eliminate the cells harboring a non-recombinant vector (non-viable clones) and to select the recombinant clones (viable clones). Example I; Construction of the plasmid PKIL19
Le gène ccdB a été amplifié par PCR en utilisant comme ADN matrice, le plasmide pULB2208 (Bernard and Couturier, The 41 carboxy-terminal residues of the iniF plasmid CcdA protein are sufficient to antagonize the killer activity of the CcdB protein, Mol. Gen. Genêt. 226, 297-304 (1991)) ainsi que des oligonucléotides synthétiques.The ccdB gene was amplified by PCR using as plasmid DNA, the plasmid pULB2208 (Bernard and Couturier, The 41 carboxy-terminal residues of the iniF plasmid CcdA protein are sufficient to antagonize the killer activity of the CcdB protein, Mol. Gen. Genet 226, 297-304 (1991)) as well as synthetic oligonucleotides.
Les séquences des oligonucléotides synthétiques ont été choisies de manière à créer un site de restriction EcoRI de part et d'autre du gène sauvage ccdB afin de pouvoir recloner ce gène en phase avec les codons des sites multiples de clonage MCS19 et d'éliminer le codon d'initiation du gène ccdB natif. L'ADN issu de la réaction PCR a été digéré par l'enzyme EcoRI et clone dans le site EcoRI du plasmide pUC19. Le plasmide résultant, ayant intégré le fragment EcoRI dans l'orientation permettant la lecture à partir du promoteur Lac du gène ccdB doté des codons additionels correspondants aux sites multiples de clonage MCS19, a été appelé pKIL2. Le plasmide pKIL2 est létal pour une bactérie sauvage (sensible CcdBs) .The sequences of the synthetic oligonucleotides were chosen so as to create an EcoRI restriction site on either side of the wild-type ccdB gene in order to be able to reclonate this gene in phase with the codons of the multiple cloning sites MCS19 and to eliminate the codon initiation of the native ccdB gene. The DNA resulting from the PCR reaction was digested with the EcoRI enzyme and cloned into the EcoRI site of the plasmid pUC19. The resulting plasmid, having integrated the EcoRI fragment in the orientation allowing the reading, from the Lac promoter, of the ccdB gene endowed with additional codons corresponding to the multiple MCS19 cloning sites, was called pKIL2. The plasmid pKIL2 is lethal for a wild bacterium (sensitive CcdB s ).
Le pKIL2 possède encore deux sites Smal, l'un au niveau des multiples sites de clonage, l'autre dans la région centrale du gène ccdB. Ce dernier a été éliminé par multagénèse site spécifique. Le plasmide résultant pKIL3, pourvu d'un unique site Smal est encore doté de deux sites EcoRI. Le site EcoRI en aval du gène ccdB a été éliminé par remplissage de ses extrémités cohésives.PKIL2 still has two SmaI sites, one at the multiple cloning sites, the other in the central region of the ccdB gene. The latter was eliminated by site-specific multagenesis. The resulting plasmid pKIL3, provided with a single SmaI site also has two EcoRI sites. The EcoRI site downstream of the ccdB gene was eliminated by filling its cohesive ends.
Le plasmide résultant, pKIL19 (figure 3) possède donc un site de restriction EcoRI unique au niveau de la séquence 5 comprenant les multiples sites de clonage. Exemple II ; construction du plasmide PKIL18The resulting plasmid, pKIL19 (FIG. 3) therefore has a unique EcoRI restriction site at the level of the sequence 5 comprising the multiple cloning sites. Example II; construction of the plasmid PKIL18
Le gène ccdB a été amplifié par PCR en utilisant comme DNA matrice, le plasmide pKIL19 ainsi que des oligonucléotides synthétiques. Les séquences des oligonucléotides synthétiques ont été choisies de manière à créer un site HindIII de part et d'autre du gène ccdB afin de pouvoir recloner ce gène en phase avec les codons des sites multiples de clonage MCS18. L'ADN issu de la réaction de PCR a été digéré par l'enzyme HindIII et cloner dans le site HindIII du plasmide pUCIS. Le plasmide résultant, ayant intégré le fragment HindIII dans l'orientation permettant la lecture à partir du promoteur Lac du gène ccdB dotés des codons additionels correspondants aux sites multiples de clonage MCS18, a été appelé pKIL4. Le plasmide pKIL4 est létal pour une bactérie sensible CcdBs.The ccdB gene was amplified by PCR using as DNA template, the plasmid pKIL19 as well as synthetic oligonucleotides. The sequences of the synthetic oligonucleotides were chosen so as to create a HindIII site on either side of the ccdB gene in order to be able to reclonate this gene in phase with the codons of the multiple MCS18 cloning sites. DNA from the reaction of PCR was digested with the enzyme HindIII and cloned into the HindIII site of the plasmid pUCIS. The resulting plasmid, having integrated the HindIII fragment in the orientation allowing the reading, from the Lac promoter, of the ccdB gene endowed with additional codons corresponding to the multiple cloning sites MCS18, was called pKIL4. The plasmid pKIL4 is lethal for a sensitive bacterium CcdB s .
Le site HindIII en aval du gène ccdB a été éliminé par remplissage de ses extrémités cohésives. Le plasmide résultant pKIL18 (figure 4), possède un site unique de restriction HindIII ainsi qu'un site Smal unique (car construit à partir du pKIL19) . Exemple III : Construction des souches CcdB1" et CcdBs The HindIII site downstream of the ccdB gene was eliminated by filling its cohesive ends. The resulting plasmid pKIL18 (FIG. 4) has a unique HindIII restriction site as well as a unique SmaI site (because it is constructed from pKIL19). Example III: Construction of the CcdB 1 " and CcdB s strains
Pour pouvoir maintenir les plasmides pKIL18 et pKIL19 au sein d'une bactérie, celle-ci doit être résistante à l'effet létal de la protéine de fusion active en tant que poison. De manière inattendue, la mutation chromosomique gyrA462 confère aux souches une résistance totale à l'effet poison de la protéine fusion. D'autre part, les plasmides pKIL18 et pKIL19 dérivant directement des plasmides pUC18 et pUC19 et exprimant les gènes ccdB à partir du promoteur Lac , il est préférable de maintenir ces plasmides dans une souche LacJq. En effet, dans notre cas une surexpression continuelle de ces gènes ne joue pas une pression de sélection en faveur de certaines mutations, mais la souche acJq permet de réduire l'expression à partir du promoteur Lac et économise la machinerie bactérienne ce qui permet de garantir un temps de génération rapide (production élevée du vecteur par la souche) .To be able to maintain the plasmids pKIL18 and pKIL19 within a bacterium, the latter must be resistant to the lethal effect of the active fusion protein as a poison. Unexpectedly, the chromosomal mutation gyrA462 gives the strains total resistance to the poison effect of the fusion protein. On the other hand, the plasmids pKIL18 and pKIL19 deriving directly from the plasmids pUC18 and pUC19 and expressing the ccdB genes from the Lac promoter, it is preferable to maintain these plasmids in a LacJ q strain. Indeed, in our case a continual overexpression of these genes does not play a selection pressure in favor of certain mutations, but the strain acJ q makes it possible to reduce the expression from the Lac promoter and saves the bacterial machinery which makes it possible to guarantee a rapid generation time (high production of the vector by the strain).
La souche D1210 (Sadler et al Gène 8, p 279-300 (1980)) issue de la souche HB101 Lacl^, LacY* (Maniatis et al (Molecular Cloning Laboratories Man. Cold Spring Harbor Laboratory N.Y.) , caractérisé par un Iq chromosomique et un taux de transformation élevé, a été transformée par le plasmide pC0S2.1. Ce plasmide, qui confère la résistance à la kanamycine, porte le gène recA d'Erwinia chrysanthemi 3665, et permet la recombinaison chez E. coli. Un lysat de phage PI a été préparé sur une souche CcdBR gyrA462, zei- 298:: TnlO et utilisé pour infecter la souche D1210/pCOS2.1. Les transductants résistants à la tétracycline ont été sélectionnés et testés pour leur résistance ou sensibilité à la protéine CcdB. L'un des transductants CcdBR a ensuite été curé du plasmide pCOS2.1 et dénommé KIB22.The strain D1210 (Sadler et al Gene 8, p 279-300 (1980)) derived from the strain HB101 Lacl ^, LacY * (Maniatis et al (Molecular Cloning Laboratories Man. Cold Spring Harbor Laboratory NY), characterized by an I q chromosome and a high transformation rate, was transformed by the plasmid pC0S2.1 This plasmid, which confers resistance to kanamycin, carries the recA gene of Erwinia chrysanthemi 3665, and allows the recombination in E. coli. of phage PI was prepared on a strain CcdB R gyrA462, zei- 298 :: TnlO and used to infect the strain D1210 / pCOS2.1. Tetracycline resistant transductants were selected and tested for their resistance or sensitivity to the CcdB protein. One of the CcdB R transductants was then cleared from the plasmid pCOS2.1 and named KIB22.
La souche KIB22 constitue une souche hôte idéale pour les plasmides pKIL18 et pKIL19 alors que la souche D1210 constitue l'hôte idéal pour la sélection des plasmides recombinants.The strain KIB22 constitutes an ideal host strain for the plasmids pKIL18 and pKIL19 while the strain D1210 constitutes the ideal host for the selection of the recombinant plasmids.
En effet, la souche KIB22 possède de manière avantageuse un taux d'extraction de l'ADN élevé (comparable au rendement des plasmides pUC) et de manière inattendue un résistance à la protéine de fusion codée par le pKILlδ et pKIL19.Indeed, the strain KIB22 advantageously has a high DNA extraction rate (comparable to the yield of plasmids pUC) and unexpectedly resistance to the fusion protein encoded by pKILlδ and pKIL19.
Par conséquent, il est possible de produire de manière industrielle par ce microorganisme, le vecteur de clonage selon l'invention en de nombreuses —copies sans occasionner la mort dudit microorganisme. La sélection se fait par simple étalement des bactéries sur milieu contenant de 1'IPTG (Isopropyl-Beta-D- thiogalacto-pyranoside) , ainsi que de 1'ampicilline.Consequently, it is possible to produce industrially by this microorganism, the cloning vector according to the invention in numerous copies without causing the death of said microorganism. The selection is made by simple spreading of the bacteria on a medium containing IPTG (Isopropyl-Beta-D-thiogalacto-pyranoside), as well as ampicillin.
La souche KIB22 a été déposée auprès du Laboratorium voor Microbiologie-Bacteriënverzameling (LMG) des Belgian Coordinated Collections of Microorganisms (BCCM) sous le n°LMG P-12601.The strain KIB22 has been deposited with the Laboratorium voor Microbiologie-Bacteriënverzameling (LMG) of the Belgian Coordinated Collections of Microorganisms (BCCM) under the number LMG P-12601.
Le vecteur de clonage pKIL19 a été déposé auprès du Laboratorium voor Moléculaire Biologie-Plasmiden Collectie (LMBP) des Belgian Coordinated Collections of Microorganisms (BCCM) sous le n° LMBP 2781.The cloning vector pKIL19 has been deposited with the Laboratorium voor Moléculaire Biologie-Plasmiden Collectie (LMBP) of the Belgian Coordinated Collections of Microorganisms (BCCM) under the number LMBP 2781.
Ces dépôts ont été fait selon les dispositions du Traité de Budapest sur la Reconnaissance Internationale du Dépôt de Microorganismes. These deposits were made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms.

Claims

REVENDICATIONS
1. Vecteur de clonage et/ou de séquençage caractérisé en qu'il comprend, incorporé dans un vecteur à réplication autonome (2) , au moins une séquence nucléotidique promotrice (3) et au moins une séquence nucléotidique (4) codant pour une protéine de fusion active en tant que poison, ladite séquence nucléotidique (4) étant obtenue par la fusion d'une séquence nucléotidique codante (5) comprenant plusieurs sites de clonage et d'une séquence nucléotidique (6) codant pour une protéine poison.1. Cloning and / or sequencing vector characterized in that it comprises, incorporated into an autonomous replicating vector (2), at least one promoter nucleotide sequence (3) and at least one nucleotide sequence (4) coding for a protein active fusion as a poison, said nucleotide sequence (4) being obtained by the fusion of a coding nucleotide sequence (5) comprising several cloning sites and a nucleotide sequence (6) coding for a poison protein.
2. Vecteur selon la revendication 1 caractérisé en ce que le vecteur à réplication autonome (2) est un virus recombinant.2. Vector according to claim 1 characterized in that the vector with autonomous replication (2) is a recombinant virus.
3. Vecteur selon la revendication 1 caractérisé en ce que le vecteur à réplication autonome (2) est un plasmide recombinant.3. Vector according to claim 1 characterized in that the vector with autonomous replication (2) is a recombinant plasmid.
4. Vecteur selon la revendication 3 caractérisé en ce que le vecteur à réplication autonome (2) est un plasmide pUC recombinant.4. Vector according to claim 3 characterized in that the vector with autonomous replication (2) is a recombinant pUC plasmid.
5. Vecteur selon l'une quelconque des revendications précédentes caractérisé en ce que la séquence nucléotidique promotrice (3) est constituée par le promoteur de 1 'opéron Lac .5. Vector according to any one of the preceding claims, characterized in that the promoter nucleotide sequence (3) consists of the promoter of the Lac operon.
6. Vecteur selon l'une quelconque des revendications précédentes caractérisé en ce que les sites uniques de clonage de la séquence nucléotidique (5) fusionnée à la séquence nucléotidique (6) codant pour la protéine poison, sont absents du reste de la séquence nucléotidique du vecteur de clonage.6. Vector according to any one of the preceding claims, characterized in that the unique cloning sites of the nucleotide sequence (5) fused to the nucleotide sequence (6) coding for the poison protein, are absent from the rest of the nucleotide sequence of the cloning vector.
7. Vecteur selon l'une ou quelconque des revendications précédentes caractérisé en ce que la séquence nucléotidique (6) codant pour une protéine poison comprend tout ou une partie de la séquence nucléotidique du gène sauvage codant pour la protéine CcdB.7. Vector according to any one of the preceding claims, characterized in that the nucleotide sequence (6) coding for a poison protein comprises all or part of the nucleotide sequence of the wild-type gene coding for the protein CcdB.
8. Vecteur selon la revendication 7 caractérisé en ce que la séquence nucléotidique (6) codant pour la protéine poison est dépourvue du site de clivage de l'enzyme de restriction Smal.8. Vector according to claim 7 characterized in that the nucleotide sequence (6) coding for the protein poison lacks the cleavage site of the restriction enzyme Smal.
9. Vecteur selon l'une quelconque des revendications précédentes caractérisé en ce qu'il correspond au dépôt n° LMBP2781.9. Vector according to any one of the preceding claims, characterized in that it corresponds to deposit No. LMBP2781.
10. Cellule procaryote transformée par le vecteur de clonage selon l'une quelconque des revendications précédentes.10. Prokaryotic cell transformed with the cloning vector according to any one of the preceding claims.
11. Cellule procaryote hôte du vecteur de clonage selon l'une quelconque des revendications précédentes 1 à 9, caractérisé en ce qu'elle comporte de préférence un Iq chromosomique , un taux de transformation élevé et comporte une mutation conférant la résistance à l'activité poison de la protéine de fusion et/ou comporte un gène codant pour une protéine antipoison de la protéine de fusion.11. Prokaryotic cell host of the cloning vector according to any one of the preceding claims 1 to 9, characterized in that it preferably comprises a chromosomal I q , a high transformation rate and comprises a mutation conferring resistance to poison activity of the fusion protein and / or comprises a gene coding for a poison protein of the fusion protein.
12. Cellule procaryote hôte du vecteur de clonage selon la revendication 11 caractérisé en ce qu'elle comporte une mutation dans le gène codant pour la sous unité A ou dans le gène codant pour la sous unité B de la gyrase et conférant la résistance à la protéine de fusion et/ou comporte un gène codant pour la protéine CcdA antipoison de la protéine de fusion.12. Prokaryotic cell host of the cloning vector according to claim 11 characterized in that it comprises a mutation in the gene coding for the subunit A or in the gene coding for the subunit B of the gyrase and conferring resistance to fusion protein and / or comprises a gene coding for the protein CcdA poison of the fusion protein.
13. Cellule d'Escherichia coli selon la revendication 11 ou 12 caractérisé en ce qu'elle comporte une mutation responsable de la substitution de l'arginine 462 par une cysteine dans la séquence des acides aminés du polypeptide GyrA de la gyrase conférant la résistance à la protéine de fusion.13. Escherichia coli cell according to claim 11 or 12 characterized in that it comprises a mutation responsible for the substitution of arginine 462 by a cysteine in the amino acid sequence of the gyrase polypeptide of gyrase conferring resistance to fusion protein.
14. Cellule selon l'une quelconque de revendications 11 à 13 caractérisé en ce qu'elle comporte la mutation LacJq.14. Cell according to any one of claims 11 to 13 characterized in that it comprises the LacJ q mutation.
15. Cellule selon l'une quelconque des revendications précédentes 11 à 14 caractérisé en ce qu'elle est déposée sous le n° LMGP-12601. 15. Cell according to any one of the preceding claims 11 to 14, characterized in that it is deposited under the number LMGP-12601.
16. Fragments du vecteur selon l'une quelconque des revendications 1 à 9, caractérisé en ce qu'ils sont constitués par des séquences de 10 à 30 nucléotides s'hybridant aux séquences situées de part et d'autre de la séquence nucléotidique (5) comprenant plusieurs sites uniques de clonage.16. Fragments of the vector according to any one of claims 1 to 9, characterized in that they consist of sequences of 10 to 30 nucleotides hybridizing to the sequences located on either side of the nucleotide sequence (5) comprising several unique cloning sites.
17. Utilisation du vecteur de clonage selon l'une quelconque des revendications 1 à 9 pour la sélection de clones recombinants. 17. Use of the cloning vector according to any one of claims 1 to 9 for the selection of recombinant clones.
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Families Citing this family (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BE1006085A3 (en) * 1992-07-31 1994-05-10 Univ Bruxelles Cloning vector.
US7176029B2 (en) 1992-07-31 2007-02-13 Universite Libre De Bruxelles Cloning and/or sequencing vector
US6720140B1 (en) 1995-06-07 2004-04-13 Invitrogen Corporation Recombinational cloning using engineered recombination sites
DE69623057T2 (en) 1995-06-07 2003-03-27 Invitrogen Corp., Carlsbad RECOMBINATORY CLONING IN VITRO USING GENE-manipulated RECOMBINATION LOCATIONS
US6143557A (en) * 1995-06-07 2000-11-07 Life Technologies, Inc. Recombination cloning using engineered recombination sites
US6964861B1 (en) 1998-11-13 2005-11-15 Invitrogen Corporation Enhanced in vitro recombinational cloning of using ribosomal proteins
US5851808A (en) 1997-02-28 1998-12-22 Baylor College Of Medicine Rapid subcloning using site-specific recombination
CN101125873A (en) * 1997-10-24 2008-02-20 茵维特罗根公司 Recombinational cloning using nucleic acids having recombination sites
ATE341621T1 (en) * 1997-10-24 2006-10-15 Invitrogen Corp RECOMBINATORY CLONING USING NUCLIC ACIDS HAVING RECOMBINATION SITE
US6022716A (en) 1998-04-10 2000-02-08 Genset Sa High throughput DNA sequencing vector
AU766257C (en) * 1998-05-07 2004-06-03 Universite Libre De Bruxelles Cytotoxin-based biological containment
NZ525134A (en) 1999-03-02 2004-09-24 Invitrogen Corp Compositions and methods for use in recombinational cloning of nucleic acids
JP2002537800A (en) * 1999-03-02 2002-11-12 インビトロゲン・コーポレーション Cells resistant to toxic genes and uses thereof
CN1757724B (en) * 1999-12-10 2014-06-11 茵维特罗根公司 Use of multiple recombination sites with unique specificity in recombinational cloning
EP1111061A1 (en) * 1999-12-20 2001-06-27 Universite Libre De Bruxelles Double selection vector
US7198924B2 (en) 2000-12-11 2007-04-03 Invitrogen Corporation Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites
US6544782B1 (en) * 2000-11-13 2003-04-08 Synthegen Systems pREM: a positive selection vector system for direct PCR cloning
CA2435956C (en) * 2001-02-23 2012-07-10 Universite Libre De Bruxelles Method for the selection of recombinant clones comprising a sequence encoding an antidote protein to toxic molecule
EP1485491A1 (en) * 2002-03-19 2004-12-15 Universite Libre De Bruxelles Poison/antidote genetic systems for the selection of genetically modified eucaryote cells or organisms
TWI320666B (en) * 2002-04-12 2010-02-11 Interdigital Tech Corp An access burst detector for use in a node b/base station
AU2003253992A1 (en) * 2002-07-18 2004-02-09 Robert P. Bennett Viral vectors containing recombination sites
JP4336770B2 (en) * 2002-07-26 2009-09-30 独立行政法人産業技術総合研究所 Markers for selection of transformants using lethal genes
WO2004022745A2 (en) * 2002-09-03 2004-03-18 Universite Libre De Bruxelles Reversible, parallel and multitask cloning method and kit
US9309518B2 (en) 2002-09-03 2016-04-12 Universite Libre De Bruxelles Reversible, parallel and multitask cloning method and kit
EP1644538A4 (en) * 2003-06-26 2006-11-08 Invitrogen Corp Methods and compositions for detecting promoter activity and expressing fusion proteins
EP1685247B1 (en) * 2003-10-03 2009-11-11 Promega Corporation Vectors for directional cloning
US8293503B2 (en) * 2003-10-03 2012-10-23 Promega Corporation Vectors for directional cloning
EP1697534B1 (en) 2003-12-01 2010-06-02 Life Technologies Corporation Nucleic acid molecules containing recombination sites and methods of using the same
EP2119789A1 (en) 2008-05-16 2009-11-18 Université Libre de Bruxelles Hyperproliferative recombinant cell
KR101077942B1 (en) 2009-02-05 2011-10-31 서울대학교산학협력단 High throughput vector for transient expression in plant and uses thereof
HUE033858T2 (en) 2012-02-14 2018-01-29 Merial Inc Rotavirus subunit vaccines and methods of making and use thereof
US10358655B1 (en) 2012-06-27 2019-07-23 University Of Utah Research Foundation Attenuated protein expression vectors and associated methods
US9333227B2 (en) 2013-08-19 2016-05-10 Syngulon Sa. Controlled growth of microorganisms
US11618899B2 (en) 2016-02-09 2023-04-04 Brookhaven Science Associates, Llc Cloning and expression vectors and systems
CN111212569A (en) 2017-08-31 2020-05-29 辛格隆股份公司 Methods and compositions for preparing bacteriocins and antimicrobial peptides
WO2019121983A1 (en) 2017-12-19 2019-06-27 Syngulon S.A. Fermentation process

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3750125T2 (en) * 1986-03-26 1994-10-27 Genexpress Aps BIOLOGICAL INSULATION.
US5464764A (en) * 1989-08-22 1995-11-07 University Of Utah Research Foundation Positive-negative selection methods and vectors
US5300431A (en) * 1991-02-26 1994-04-05 E. I. Du Pont De Nemours And Company Positive selection vector for the bacteriophage P1 cloning system
US7176029B2 (en) * 1992-07-31 2007-02-13 Universite Libre De Bruxelles Cloning and/or sequencing vector
BE1006085A3 (en) 1992-07-31 1994-05-10 Univ Bruxelles Cloning vector.
JP2590761B2 (en) * 1994-11-22 1997-03-12 日本電気株式会社 TAB semiconductor device and method of connecting TAB semiconductor device to circuit board
US6143557A (en) * 1995-06-07 2000-11-07 Life Technologies, Inc. Recombination cloning using engineered recombination sites
DE69623057T2 (en) * 1995-06-07 2003-03-27 Invitrogen Corp., Carlsbad RECOMBINATORY CLONING IN VITRO USING GENE-manipulated RECOMBINATION LOCATIONS
US5922583A (en) * 1995-10-17 1999-07-13 Biostar Inc. Methods for production of recombinant plasmids
AU766257C (en) 1998-05-07 2004-06-03 Universite Libre De Bruxelles Cytotoxin-based biological containment
US6271359B1 (en) * 1999-04-14 2001-08-07 Musc Foundation For Research Development Tissue-specific and pathogen-specific toxic agents and ribozymes
CA2435956C (en) * 2001-02-23 2012-07-10 Universite Libre De Bruxelles Method for the selection of recombinant clones comprising a sequence encoding an antidote protein to toxic molecule
EP1485491A1 (en) * 2002-03-19 2004-12-15 Universite Libre De Bruxelles Poison/antidote genetic systems for the selection of genetically modified eucaryote cells or organisms

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9403616A3 *

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